Human cervical cancer 2 protooncogene and protein encoded therein

ABSTRACT

A human cervical cancer 2 protooncogene having a base sequence of SEQ ID: 1 or a fragment thereof is overexpressed in various cancer tissues and can be used in diagnosing various cancers and an anti-sense gene complementary thereto can be used in treating cancers.

This application is a national stage 371 application of PCT/KR01/01172,filed on Jul. 9, 2001, which claims benefit from application Republic ofKorea 2000-71202 filed on Nov. 28, 2000.

FIELD OF THE INVENTION

The present invention relates to a novel protooncogene and proteinencoded therein, and more particularly, to a human cervical cancer 2protooncogene and a protein derived therefrom, which can be used indiagnosis of various cancers.

BACKGROUND OF THE INVENTION

Higher animals including man each carry approximately 100,000 genes, butonly about 15% thereof is expressed, and characteristics of individual'sbiological process, e.g., genesis, differentiation, homeostasis,responses to stimuli, control of cell segmentation, aging andapoptosis(programmed cell death), are determined depending on whichgenes are expressed(see Liang, P. and A. B. Pardee, Science, 257:967-971(1992)).

Pathogenic phenomena such as tumorigenesis are caused by gene mutationwhich brings about changes in the mode of gene expression. Therefore,comparative studies of gene expressions in various cells have beenconducted to provide bases for establishing viable approaches to theunderstanding of diverse biological phenomena.

For example, the mRNA differential display(DD) method suggested by Liangand Pardee is effective in elucidating the nature of tumor suppressorgenes, cell cycle-related genes and transcriptional regulatory genesthat control apoptosis(see Liang, P. and A. B. Pardee supra). Further,the DD method has been widely used in examining the interrelationship ofvarious genes in a cell.

It has been reported that tumorigenesis is caused by various geneticchanges such as the loss of chromosomal heterozygosity, activation ofoncogenes and inactivation of tumor suppressor genes, e.g., p53 gene(seeBishop, J. M., Cell, 64: 235-248(1991); and Hunter, T., Cell, 64:249-270(1991)). Further, it has been reported that 10 to 30% of humancancer arises from the activation of oncogene through amplification ofprotooncogenes.

Therefore, the activation of protooncogenes plays an important role inthe etiology of many tumors and there has existed a need to identifyprotooncogenes.

The present inventor has endeavored to unravel examine the mechanisminvolved in the tumorigenesis of cervical cancer; and, has unexpectedlyfound that a novel protooncogene, human cervical cancer 2(HCCR-2), isspecifically overexpressed in cancer cells. This protooncogene can beeffectively used in diagnosis, prevention and treatment of variouscancers, e.g., leukemia, lymphoma, colon, breast, kidney, stomach, lung,ovary and uterine cervix cancers.

SUMMARY OF THE INVENTION

Accordingly, the primary object of the present invention is to provide anovel protoncogene and a fragment thereof.

Other objects of the present invention are to provide:

a recombinant vector containing said protooncogene or a fragment thereofand a microorganism transformed therewith;

a protein encoded in said protooncogene and a fragment thereof;

a kit for diagnosis of cancer containing said protooncogene or afragment thereof;

a kit for diagnosis of cancer containing said protein or a fragmentthereof;

an anti-sense gene having a base sequence complementary to that of saidprotooncogene or a fragment thereof; and

a process for treating or preventing cancer by using said anti-sensegene.

In accordance with one aspect of the present invention, there isprovided a novel protooncogene having the nucleotide sequence of SEQ IDNo:1 or a fragment thereof.

In accordance with another aspect of the present invention, there isprovided a recombinant vector containing said protooncogene or afragment thereof and a microorganism transformed with said vector.

In accordance with still another aspect of the present invention, thereis provided a protein having the amino acid sequence of SEQ ID No:2 or afragment thereof derived from said protooncogene or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings which respectivelyshow;

FIG. 1: the DD identification of altered gene expression in normalcervix tissue, primary cervical cancer tissue, metastatic lymph nodetissue and cervical cancer cell line (CUMC-6);

FIG. 2: the results of northern blot analyses for HCCR-2 gene expressedin normal cervical tissues, cervical cancer tissues and cervical cancercell lines(CaSki and CUMC-6);

FIG. 3A: the results of northern blot analyses for HCCR-2 gene expressedin normal human 12-lane multiple tissues;

FIG. 3B: the results obtained with the same sample of FIG. 3A hybridizedwith β-actin;

FIG. 4A: the results of northern blot analyses for HCCR-2 gene expressedin human cancer cell lines;

FIG. 4B: the results obtained with the same sample of FIG. 4A hybridizedwith β-actin;

FIG. 5A: the results of northern blot analyses for HCCR-2 gene expressedin human tumor tissues and their normal counterparts;

FIG. 5B: the results obtained with the same sample of FIG. 5A hybridizedwith β-actin;

FIG. 6: a phase-contrast feature of monolayer-cultured wild type NIH/3T3cells;

FIG. 7: a phase-contrast feature of monolayer-culturedHCCR-2-transfected NIH/3T3 cells (HCCR-2M cells);

FIG. 8: hematoxylin-eosin staining of monolayer-culturedHCCR-2-transfected NIH/3T3 cells;

FIG. 9: tumorigenicity of HCCR-2-transfected NIH/3T3 cells in nudemouse;

FIG. 10: hematoxylin-eosin staining of subcutaneous tumor nodulesderived from HCCR-2-transfected NIH/3T3 cells in nude mice;

FIG. 11: phase-contrast features of monolayer-cultured nude mice-derivedHCCR-2MN cells;

FIG. 12 sodium dodecyl sulfate (SDS)-PAGE results showing proteinexpression patterns before and after the IPTG induction;

FIG. 13: the result of western blotting analysis of NIH/3T3 cellswithout transfection(wild type), NIH/3T3 transfected with pcDNA3 vectoralone(pcDNA3) and HCCR-2-transfected NIH/3T3 cells (HCCR-2M cells);

FIG. 14: the result of western blotting analysis of human tumor tissuesof kidney, breast, lung, ovary and stomach and their normalcounterparts;

FIGS. 15A and 15B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal leukocytes and human leukemia cells(×200);

FIGS. 16A and 16B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal lymphocytes and human lymphoma tissue(×200);

FIGS. 17A and 17B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal breast tissue and human breast cancer(×200);

FIGS. 18A and 18B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal colon tissue and human colon cancer(×100);

FIGS. 19A and 19B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal kidney tissue and human kidney tumortissue (×100);

FIGS. 20A and 20B: the results of immunohistochemical stainings forHCCR-2 protein expressed in normal cervical tissue and human cervicalcancer tissue (×100);

FIG. 21: phase-contrast features of monolayer-cultured wild type humanembryonic kidney 293 epithelial cells;

FIG. 22: phase-contrast features of monolayer-culturedHCCR-2-transfected 293 epithelial cells (HCCR-2H cells);

FIG. 23: hematoxylin-eosin staining of monolayer-culturedHCCR-2-transfected 293 epithelial cells;

FIG. 24: tumorigenicity of HCCR-2-transfected 293 epithelial cells innude mouse;

FIG. 25: hematoxylin-eosin staining of subcutaneous tumor nodulesderived from HCCR-2-transfected 293 epithelial cells in nude mice;

FIG. 26: phase-contrast features of monolayer-cultured nude mice-derivedHCCR-2HN cells; and

FIG. 27: the results of northern blot analyses for wild type 293epithelial cell and HCCR-2-transfected 293 epithelial cells (clones A,B, C, D and E).

DETAILED DESCRIPTION OF THE INVENTION

The novel protooncogene of the present invention, i.e., human cervicalcancer 2(hereinafter “HCCR-2 protooncogene”), consists of 2003 basepairs and has the DNA sequence of SEQ ID NO:1.

In SEQ ID NO: 1, the full open reading frame corresponding to base Nos.63 to 977 is a protein encoding region and the predicted amino acidsequence derived therefrom is shown in SEQ ID NO: 2 which consists of304 amino acids(“HCCR-2 protein”). Further, the region represented bynucleotide No. 321 to 380 of SEQ ID NO: 1 encodes a single transmembranedomain having the predicted amino acid sequence of amino acid Nos. 87 to106 of SEQ ID NO: 2. This suggests that the protooncogene of the presentinvention is a membrane-bound gene.

A single potential N-glycosylation site(corresponding to base Nos. 831to 839 of SEQ ID NO: 1 and amino acid Nos. 257 to 259 of SEQ ID NO: 2)is present at the C-terminal side of the HCCR-2 protein, which suggeststhat HCCR-2 is a type II membrane protein. The polyadenylation signalcorresponds to the nucleotide Nos. 1894-1898 of SEQ ID NO: 1.

In consideration of the degeneracies of codons and the preferred codonsin a specific animal wherein the protooncogene of the present inventionis to be expressed, various changes and modifications of the DNAsequences of SEQ ID NO:1 may be made, e.g., in the coding area thereofwithout adversely altering the amino acid sequence of the expressedprotein, or in the non-coding area without adversely affecting theexpression of the protooncogene. Therefore, the present invention alsoincludes, in its scope, a polynucleotide having substantially the samebase sequence as the inventive protooncogene, and a fragment thereof. Asused herein, “substantially the same polynucleotide” refers to apolynucleotide whose base sequence shows 80% or more, preferably 90% ormore, most preferably 95% or more homology to the protooncogene of thepresent invention.

The protein expressed from the protooncogene of the present inventionconsists of 304 amino acids and has the amino acid sequence of SEQ IDNO: 2. The molecular weight of this protein is about 45 kDa. However,various substitution, addition and/or deletion of the amino acidresidues of protein may be performed without adversely affecting theprotein's function. Further, a portion of the protein may be used when aspecific purpose is to be fulfilled. These modified amino acids andfragments thereof are also included in the scope of the presentinvention. Therefore, the present invention includes, in its scope, apolypeptide having substantially the same amino acid sequence as theprotein derived from the oncogene of the present invention and afragment thereof. As used herein, “substantially the same polypeptide”refers to a polypeptide whose amino acid sequence shows 80% or more,preferably 90% or more, most preferably 95% or more homology to theamino acid sequence of SEQ ID NO: 2.

The protooncogene, or the protein, of the present invention can beobtained from human cancer tissues or synthesized using a conventionalDNA or peptide synthesis method. Further, the gene thus prepared may beinserted to a conventional vector to obtain an expression vector, whichmay, in turn, be introduced into a suitable host, e.g., a microorganismsuch as an E. coli or yeast, or an animal cell such as a mouse or humancell.

The transformed host may then be used in producing the inventive DNA orprotein on a large scale. For example, E. coli JM109 is transformed withexpression vector pCEV-LAC (Miki, T. et al., Gene, 83: 137-146 (1989))containing the inventive HCCR-2 gene (designated HCCR-2/pCEV-LAC) toobtain an E. coli transformant designated JM109/HCCR2 which wasdeposited on Oct. 11, 1999 with the Korean Collection for TypeCultures(KCTC)(Address: Korea Research Institute of Bioscience andBiotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333,Republic of Korea) under the accession number, KCTC 0668BP, inaccordance with the terms of Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

In preparing a vector, expression-control sequences, e.g., promoter.terminator, self replication sequence and secretion signal, are suitablyselected depending on the host cell used.

The overexpression of the protooncogene of the present invention occursnot in normal cervical tissue but in cervical cancer tissues andcervical cancer cell lines. This suggests that the protooncogene of thepresent invention induces cervical cancer. Further, when a normalfibroblast cell, e.g., NIH/3T3 cell line, is transfected with theprotooncogene of the present invention, an abnormal cells is produced.Morphological characterizations with optical and electronic microscopesshow that the abnormal cell has the form of a tumor cell.

When the normal fibroblast cell transfected with the protooncogene ofthe present invention is injected into the posterial lateral aspect of anude mouse, tumorigenesis is observed after about 21 days from theinjection, the tumor size becoming 1 cm×1 cm in 40 days. By usinghematoxylin-eosin dye method, it can be confirmed that the tumor cellsare cancerous.

In addition to epithelial tissues such as cervical cancer tissue, theoverexpression of the protooncogene of the present invention is alsoobserved in various other cancer tumors such as leukemia, lymphoma,breast, kidney, ovary, lung and stomach cancers. Therefore, theprotooncogene of the present invention is believed to be a factor commonto all forms of various cancer and it can be advantageously used in thediagnosis of various cancers and the production of a transformed animalas well as in an anti-sense gene therapy.

A diagnostic method that can be performed using the protooncogene of thepresent invention may comprise, for example, the steps of hybridizingnucleic acids separated from the body fluid of a subject with a probecontaining the protooncogene of the present invention or a fragmentthereof, and determining whether the subject has the protooncogene byusing a conventional detection method in the art. The presence of theprotooncogene may be easily detected by labeling the probe with aradioisotope or an enzyme. Therefore, a cancer diagnostic kit containingthe protooncogene of the present invention or a fragment thereof is alsoincluded in the scope of the present invention.

A transformed animal produced by introducing the protooncogene of thepresent invention into a mammal, e.g., a rat, is also included in thescope of the present invention. In producing such a transformed animal,it is preferred to introduce the inventive protooncogene to a fertilizedegg of an animal before the 8th cell cycle stage. The transformed animalcan be advantageously used in screening for carcinogens or anticanceragents such as antioxidants.

The present invention also provides an anti-sense gene which is usefulin a gene therapy. As used herein, the term “an anti-sense gene” means apolynucleotide comprising a base sequence which is fully or partiallycomplementary to the sequence of the mRNA which is transcribed from theprotooncogene having the base sequence of SEQ ID NO: 1 or a fragmentthereof, said nucleotide being capable of preventing the expression ofthe open reading frame(ORF) of the protooncogene by way of attachingitself to the protein-binding site of mRNA.

The present invention also includes within its scope a process fortreating or preventing cancer in a subject by way of administering atherapeutically effective amount of the inventive anti-sense genethereto.

In the inventive anti-sense gene therapy, the anti-sense gene of thepresent invention is administered to a subject in a conventional mannerto prevent the expression of the protooncogene. For example, theanti-sense ODN is mixed with a hydrophobized poly-L-lysine derivative byelectrostatic interaction in accordance with the method disclosed byKim, J. S. et al.(J. Controlled Release, 53: 175-182(1998)) and theresulting mixed anti-sense ODN is administered intravenously to asubject.

The present invention also includes within its scope an anti-cancercomposition comprising the anti-sense gene of the present invention asan active ingredient, in association with pharmaceutically acceptablecarriers, excipients or other additives, if necessary. Thepharmaceutical composition of the present invention is preferablyformulated for administration by injection.

The amount of the anti-sense gene actually administered should bedetermined in light of various relevant factors including the conditionto be treated, the chosen route of administration, the age and weight ofthe individual patient, and the severity of the patient's symptoms.

The protein expressed from the inventive protooncogene may be used inproducing an antibody useful as a diagnostic tool. The antibody of thepresent invention may be prepared in the form of a monoclonal orpolyclonal antibody in accordance with any of the methods well known inthe art by using a protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof. Cancer diagnosis may be carried out using anyof the methods known in the art, e.g., enzyme linkedimmunosorbentassay(ELISA), radioimmunoassay(RIA), sandwich assay,immunohistochemical staining, western blot or immunoassay blot onpolyacrylic gel, to asses whether the protein is expressed in the bodyfluid of the subject. Therefore, a cancer diagnostic kit containing theprotein having the amino acid sequence of SEQ ID NO: 2 or a fragmentthereof is also included in the scope of the present invention.

A continuously viable cancer cell line may be established by using theprotooncogene of the present invention, and such a cell line may beobtained, for example, from tumor tissues formed on the back of a nudemouse by injecting fibroblast cells transformed with the protooncogeneof the present invention. The cell lines thus prepared may beadvantageously used in searching for anti-cancer agents.

The following Examples and Test Examples are given for the purpose ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1 Differential Display of mRNA

(Step 1) Isolation of Total RNA

Normal exocervical tissue specimens were obtained from uterine myomapatients during hysterectomy, and untreated primary cervical cancer andmetastatic common iliac lymph node tissue specimens were obtained duringradical hysterectomy. The human cervical cancer cell line CUMC-6 (Kim JW et al., Gynecol Oncol, 62: 230-240 (1996)) was cultured in Waymouth MB751/1 medium.

Total RNAs were extracted from the tissue specimens and cells using acommercial system (RNeasy total RNA kit, Qiagen Inc., Germany), and DNAcontaminants were removed therefrom using Message clean kit (GenHunterCorp., Brookline, Mass.).

(Step 2) Differential Display

Differential display was conducted according to Liang et al. (Science,257: 967-971 (1992); and Cancer Res., 52: 6966-6968 (1992)) with minormodifications as follows.

0.2 μg each of the total RNAs obtained in Step 1 was subjected toreverse transcription using primer of SEQ ID NO: 3, as an anchoredoligo-dT primer (RNAimage kit, GenHunter), followed by polymerase chainreaction (PCR) using the same anchored primer and the arbitrary 5′ 13mer (RNAimage primer set 1, H-AP 1-32) in the presence of 0.5 mM[α-³⁵S]-labeled dATP (1200 Ci/mmol). The PCR thermal cycle was repeated40 times, each cycle being composed of: 95° C. for 40 sec., 40° C. for 2min. and 72° C. for 40 sec., and finally the reaction was carried out at72° C. for 5 min. The PCR product thus obtained was subjected toelectrophoresis in 6% polyacrylamide sequencing gels, followed byautoradiography.

FIG. 1 shows the differential display results of normal exocervicaltissue, cervical cancer tissue, metastatic tissue and cervical cancercell line CUMC-6 using the anchored oligo-dT primer H-T11C(SEQ ID NO: 5)and the arbitrary 5′ 13 mer of SEQ ID NO: 3 wherein the arrow indicatesa 206 bp fragment, designated CC214, expressed in the cervical cancerand metastatic common iliac lymph node tissue, and human cervical cancercell line CUMC-6.

The band of fragment CC214 was excised from the dried sequencing gel andboiled in water for 15 min. to elute fragment CC214. The fragment CC214was subjected to PCR using the same conditions except that[α-³⁵S]-labeled DATP and 20 μM dNTPs were omitted. The amplifiedfragment CC214 was cloned into the pGEM-T Easy vector using the TACloning System (Promega, USA) and its nucleotide sequence was determinedusing the Sequenase Version 2.0 DNA Sequencing System (United StatesBiochemical Co., USA). Comparative analysis of the nucleotide sequenceof fragment CC214 with GenBank database was conducted using BLAST andFASTA programs and the result showed that this fragment has littlesequence similarity to any nucleotide sequence registered in the GenBankdatabase.

EXAMPLE 2 cDNA Library Screening

A bacteriophage λgt11 human lung embryonic fibroblast cDNA library(generously provided by Prof. I Y Chung at Hanyang University, Seoul,Korea) was screened by plaque hybridization with ³²P-labeledrandom-primed CC214 cDNA probe (Sambrook, J. et la., Molecular Cloning:A laboratory manual, New York: Cold Spring Harbor Laboratory (1989)) toobtain a full-length cDNA clone (designated HCCR-2). The nucleotidesequence of the full-length HCCR-2 cDNA clone was determined.

The full-length HCCR-2 cDNA clone contains a 2003 bp insert having thenucleotide sequence of SEQ ID NO: 1 and a full open reading framecorresponding to base Nos. 63 to 977 is a protein encoding region andthe predicted amino acid sequence derived therefrom is shown in SEQ IDNO: 2 which consists of 304 amino acids. Further, the region representedby nucleotide No. 321 to 380 of SEQ ID NO: 1 encodes a singletransmembrane domain having the predicted amino acid sequence of aminoacid Nos. 87 to 106 of SEQ ID NO: 2. This suggests that theprotooncogene of the present invention is a membrane-bound gene.

A single potential N-glycosylation site(corresponding to base Nos. 831to 839 of SEQ ID NO: 1 and amino acid Nos. 257 to 259 of SEQ ID NO: 2)is present at the C-terminal side of the HCCR-2 protein, which suggeststhat HCCR-2 is a type II membrane protein. The polyadenylation signalcorresponds to the nucleotide Nos. 1894-1898 of SEQ ID NO:1.

The nucleotide sequence of full-length HCCR-2 cDNA clone was registeredat GenBank as accession no. AF315598.

The full-length HCCR-2 cDNA was inserted in vector pCEV-LAC (Miki, T. etal., Gene, 83: 137-146 (1989)) to obtain the recombinant vectorHCCR-2/pCEV-LAC and E. coli JM109 was transformed with the recombinantvector HCCR-2/pCEV-LAC to obtain the transformed E. coli designatedJM109/HCCR2 which was deposited with Korean Collection for Type Cultures(Address: #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea)on Oct. 11, 1999 under the accession number of KCTC 0668BP.

EXAMPLE 3 Northern Blot Analysis

To determine the expression level of HCCR-2 gene in various normaltissues, cancer tissues and cancer cell lines, the northern blotanalysis was conducted as follows.

Total RNAs were prepared from normal exocervical tissue, primarycervical cancer tissue; and human cervical cancer cell lines CaSki (ATCCCRL 1550) and CUMC-6 by repeating the procedure of Step 1 of Example 1.20 μg each of the total RNAs were denatured and then electrophoresedthrough 1% formaldehyde agarose gel and transferred to nylon membranes(Boehringer-Mannheim, Germany). The blots were hybridized overnight at42° C. with ³²P-labeled random-primed HCCR-2 cDNA probe which wasprepared using a rediprime II random prime labeling system (Amersham,England). The northern blot analysis results were consistently repeatedtwo times, as quantified by densitometry and the same blots werehybridized with a β-actin probe to confirm mRNA integrity.

Using normal human 12 multiple-tissues (Clontech) and human cancer celllines (Clontech), northern blot analyses were also carried out asrecommended by the supplier.

FIG. 2 shows the northern blot analysis results of normal cervicaltissues, primary cervical cancer tissues and cervical cancer cell linesCUMC-6 and CaSki using the HCCS-1 cDNA probe; and the same blothybridized with a β-actin probe. As can be seen from FIG. 2, theexpression level of HCCR-2 gene was elevated in the cervical cancertissues and the cervical cancer cell lines but nearly absent in allnormal cervical tissues.

FIG. 3A shows the northern blot analysis results of normal humantissues, i.e., brain, heart, skeletal muscle, colon, thymus, spleen,kidney, liver, small intestine, placenta, lung and peripheral bloodleukocyte using HCCR-2 cDNA probe; and FIG. 3B, the same blot hybridizedwith a β-actin probe. As can be seen in FIG. 3A, HCCR-2 mRNA (˜2.0 kb)is weakly present or absent in many normal tissues, but the level ofexpression was high in normal kidney tissue.

FIG. 4A shows the northern blot analysis for HCCR-2 gene expressed inresults of human leukemia and lymphoma cell lines, i.e., promyelocyticleukemia HL-60 cell, HeLa cervical cancer cell, chronic myelogenousleukemia K-562 cell, lymphoblastic leukemia MOLT-4 cell, Burkitt'slymphoma Raji cell, SW480 colon cancer cell, A549 lung cancer cell andG361 melanoma cell using the HCCR-2 cDNA probe; and FIG. 4B, the sameblot hybridized with a β-actin probe. As can be seen in FIGS. 4A and 4B,HCCR-2 is transcribed at a high level in the human leukaemia andlymphoma cell lines such as chronic myelogenous leukaemia K-562,Burkitt's lymphoma Raji, lymphoblastic leukaemia MOLT-4 andpromyelocytic leukaemia HL-60 as well as HeLa cells.

K-562, MOLT-4 and HL-60, in particular, show higher transcription levelsas compared with normal leukocyte by factors of approximately 190, 90and 70, respectively. HCCR-2 expression levels in colorectal cancerSW480, lung cancer A549 and melanoma G361 cell lines are lower thanthose of leukemia and lymphoma.

Further, northern blotting analyses of the human kidney, breast, lung,ovary and stomach cancer tissues and their normal counterparts werecarried out. As shown in FIG. 5A, HCCR-2 was transcribed at a high levelin the human cancer cells, while the expression of HCCR-2 gene is barelyobservable in the normal cells. FIG. 5B shows the results obtained withthe same samples hybridized with β-actin probe to confirm mRNAingegrity.

EXAMPLE 4 Construction of Expression Vectors and Transformation ofAnimal Cells

(Step 1) Preparation of a Vector Containing HCCR-2

An expression vector containing the coding region of HCCR-2 wasconstructed as follows.

First, the entire HCCR-2 cDNA obtained in Example 2 was inserted intothe SalI restriction site of a prokaryotic expression vector,pCEV-LAC(see Miki, T. et al., Gene, 83: 137-146 (1989)). Then, the SalIfragment was isolated from the pCEV-LAC/HCCR-2 vector.

Then, pcDNA3 (Invitrogen) was digested with XhoI to make a compatibleend with SalI. The SalI fragment containing the full length HCCR-2coding sequence was inserted into the XhoI-digested pcDNA3.Lipofectamine (Gibco BRL) was used to introduce the resultingpcDNA3/HCCR-2 expression vector into NIH/3T3 cells(ACTC CRL, 1658, USA),followed by selection in a medium supplemented with G418 (Gibco). Theresulting NIH/3T3 cells transfected with HCCR-2 was designated “HCCR-2Mcells”. Another population of NIH/3T3 cells containing pcDNA3 alone wasprepared as a control and designated “pcDNA3 cells”.

(Step 2) NIH13T3 Fibroblast Cells Transfected with the HCCR-2Protooncogene

The wild type normal NIH/3T3 cell, a differentiated fibroblast cellline, is a spindle shaped cell having a long slender nucleus and ascanty amount of cytoplasm as shown in FIG. 6. When HCCR-2 was expressedin the NIH/3T3 expressing HCCR-2 (HCCR-2M cells) obtained in Step 1, thecell shape changes into a polygonal form with an ovoid nucleus and plumpcytoplasm, as shown in FIG. 7.

Monolayer cultured HCCR-2-transfected NIH/3T3 cells which is stainedwith hematoxylin-eosin, exhibit nuclear pleiomorphism, distinctnucleoli, granular chromatin patterns, tumor giant cells and atypicalmitotic figures as shown in FIG. 8.

EXAMPLE 5 Tumorigenicity of HCCR-2M Cell in Animal

To analyze tumourigenicity, 5×10⁶ HCCR-2-transfected NIH/3T3cells(HCCR-2M cells) were injected subcutaneously into the posteriorlateral aspect of the trunk of 10 mice (5-week-old athymic nu/nu onBALB/c background). Nude mice were sacrificed when the subcutaneoustumors reached 1.5-2.5 cm in diameter.

All 10 mice injected with HCCR-2M cells showed palpable tumors after 21days as shown in FIG. 9.

Nude mice bearing HCCR-2M allografts display characteristics of anepithelial carcinoma. FIG. 10 shows hematoxylin-eosin staining ofsubcutaneous tumor nodules taken from the nude mice. The sections of thetumor nodules revealed typical epithelial cell nests separated byfibrous stroma.

EXAMPLE 6 Establishment of New Cancer Cell Line from HCCR-2MCell-Induced Tumor Tissue

The cells obtained from the tumor tissue of Example 5 was cultured in aconventional manner using 20% fetal bovine serum and the cultured cellswere designated HCCR-2MN cells which have cytological features similarto HCCR-2M cells in vitro as shown in FIG. 11.

EXAMPLE 7 Determination of Size of Protein Expressed after theTransfection of E. coli with HCCR-2 Protooncogene

A full-length HCCR-2 protooncogene of SEQ ID NO: 1 was inserted into themultiple cloning site of pET-32b(+) vector(Novagen) and the resultingpET-32b(+)/HCCR-2 vector was transfected into E. coli BL21(ATCC 47092).The transfected E. coli was incubated using an LB broth medium in arotary shaking incubator, diluted by {fraction (1/100)}, and incubatedfor 3 hours. 1 mM isopropyl β-D-thiogalacto-pyranoside(IPTG, Sigma) wasadded thereto to induce the protein synthesis.

The E. coli cells in the culture were disrupted by sonication andsubjected to gel electrophoresis using 12% sodium dodecyl sulfate(SDS)before and after the IPTG induction. FIG. 12 shows the SDS-PAGE resultswhich exhibit a protein expression pattern of the E. coli BL21 straintransfected with pET-32b(+)/HCCR-2 vector. After the IPTG induction, asignificant protein band was observed at about 45 kDa. This 45 kDa fusedprotein contained an about 20 kDa Trix-Tag thioredoxin protein expresseda the gene in pET-32b(+) vector.

EXAMPLE 8 Production of Antibody

The 45 kDa fused protein isolated from the E. coli BL21 straintransfected with pET-32b(+)/HCCR-2 vector in Example 7 was purified byusing a His-Bind Kit (Novagen). Immunoblotting of the purified peptideconfirmed the presence of a major amount of a 45 kDa protein.

Then, two 6-seek old Sprague-Dawley rats each weighing about 150 g wereeach subcutaneously immunized with 1 mg of the peptide thus obtained,weekly for 3 times. Blood samples were obtained from the immunized ratsand centrifuged to obtain a polyclonal serum. The anti-HCCR-2 activityof the polyclonal serum was determined and confirmed by enzyme-linkedimmunosorbent assay(1:10,000)

EXAMPLE 9 Immunoblot Confirming Antibody Specificity

For western blot analysis, the cell identified in FIG. 7 was harvestedand lysed in a Laemmli sample buffer in accordance with the methoddescribed by Laemmli (Nature, 227: 680-685(1970)). The cellular proteinwas separated by 10% SDS-PAGE and then electroblotted ontonitrocellulose membranes. The membranes were incubated with the ratpolyclonal anti-HCCR-2 serum prepared in Example 8 for 16 h. Afterwashing, the membranes were incubated with a blocking solutioncontaining 1:1,000 dilution of peroxidase-conjugated goat anti-ratimmunoglobulin (Jackson ImmunoResearch) as a secondary antibody.Proteins were revealed by an ECL-Western blot detection kit (Amersham).

As shown in FIG. 13, HCCR-2 protein is overexpressed in HCCR-2 cells,while only faint bands are observed for the wild type and cellstransfected with the vector alone(pcDNA3). This result illustrates thespecificity of the anti-HCCR-2 antibodies in the polyclonal serum.

Further, the HCCR-2 antibody in the polyclonal serum recognizedapproximately 45 kDa protein in human protein extracts from differenttissues. As shown in FIG. 14, human tumor tissues including carcinomasof the kidney, breast, lung, ovary and stomach showed increased HCCR-2protein expression when compared with their normal counterparts.

EXAMPLE 10 Immunohistochemical Staining

To determine the expression level of HCCR-2 protein in various normaltissues and cancer tissues, the immunohistochemical stainings wereconducted according to the avidin-biotin-peroxidase complex method (Hsu,S. M. et al., Histochem Cytochem, 29: 577-580 (1981)) as follows.

The paraffin-embedded tissues were dewaxed using xylene, treated withgraded ethanol, rehydrated after removing paraffin and washed withwater. Then, the tissues were placed in peroxide quenching solution madewith hydrogen peroxide for 30 minutes to remove the endogenousperoxidase activity, treated with serum blocking solution (ZymedLaboratories, CA, USA) for 30 minutes to block non-specific binding,treated with the primary antibody, left for overnight at 4° C., washedwith phosphate-buffered saline (PBS), treated with the biotinylatedsecondary antibody (Zymed) for 30 minutes, and washed with PBS. Then,the tissues were treated with an enzyme conjugate for 30 minutes, washedwith PBS, treated with the aminoethyl carbazole (Zymed) as a chromogenfor 18 minutes. Then, the tissue sections were counterstained withhematoxylin.

FIG. 15 shows the immunohistochemical staining results of normalleukocytes(A) and leukemic cells(B). The normal leukocytes show negativeintensity of expression and leukemic cells show strong positiveintensity of expression.

FIG. 16 shows the immunohistochemical staining results of normallymphocyte(A) and lymphoma tissue(B). The normal lymphocytes shownegative intensity of expression and lymphoma tissue shows strongpositive intensity of expression in the cytoplasm.

FIG. 17 shows the immunohistochemical staining results of normal breasttissue(A) and breast tumor tissue(B). The normal breast tissue showsnegative intensity of expression and breast tumor tissue shows strongpositive intensity of expression in the cytoplasm.

FIG. 18 shows the immunohistochemical staining results of normal colontissue(A) and colon cancer tissue(B). The normal colon tissue showsnegative intensity of expression and colon cancer tissue shows strongpositive intensity of expression in the cytoplasm.

FIG. 19 shows the immunohistochemical staining results of normal kidneytissue(A) and renal cancer tissue(B). The normal kidney tissue showsnegative intensity of expression and kidney tumor tissue shows strongpositive intensity of expression in the cytoplasm.

FIG. 20 shows the immunohistochemical staining results of normalcervical tissue(A) and cervical cancer tissue(B). The portion of normalcervical tissue shows negative intensity of expression and cervicalcancer tissue shows strong positive intensity of expression.

EXAMPLE 11 Construction of Expression Vectors and Transformation ofHuman Cells

(Step 1) Preparation of a Vector Containing HCCR-2

An expression vector containing the coding region of HCCR-2 wasconstructed as follows.

First, the entire HCCR-2 cDNA obtained in Example 2 was inserted intothe SalI restriction site of a prokaryotic expression vector,pCEV-LAC(see Miki, T. et al., Gene, 83: 137-146 (1989)). Then, the SalIfragment was isolated from the pCEV-LAC/HCCR-2 vector.

Then, pcDNA3 (Invitrogen) was digested with XhoI to make a compatibleend with SalI. The SalI fragment containing the full length HCCR-2coding sequence was inserted into the XhoI-digested pcDNA3.Lipofectamine (Gibco BRL) was used to introduce the resultingpcDNA3/HCCR-2 expression vector into human embryonic kidney 293epithelial cells(ACTC CRL, 1573, USA), followed by selection in a mediumsupplemented with G418 (Gibco). The resulting 293 epithelial cellstransfected with HCCR-2 was designated “HCCR-2H cells”.

(Step 2) 293 Epithelial Cells Transfected with the HCCR-2 Protooncogene

The wild type human embryonic kidney 293 epithelial cell, adifferentiated fibroblast cell line, is a spindle shaped cell having along slender nucleus and a scanty amount of cytoplasm as shown in FIG.21. When HCCR-2 was expressed in the 293 epithelial cell expressingHCCR-2(HCCR-2H cells) obtained in Step 1, the cell shape changes into apolygonal form with an ovoid nucleus and plump cytoplasm, as shown inFIG. 22.

Monolayer cultured HCCR-2-transfected 293 epithelial cells which arestained with hematoxylin-eosin, exhibit nuclear pleiomorphism, distinctnucleoli, granular chromatin patterns, tumor giant cells and atypicalmitotic figures as shown in FIG. 23.

EXAMPLE 12 Tumorigenicity of HCCR-2H Protooncogene in Animal

To analyze tumourigenicity, 5×10⁶ HCCR-2-transfected human embryonickidney 293 epithelial cells(HCCR-2H cells) were injected subcutaneouslyinto the posterior lateral aspect of the trunk of 10 mice (5-week-oldathymic nu/nu on BALB/c background). Nude mice were sacrificed when thesubcutaneous tumors reached 1.5-2.5 cm in diameter.

All 10 mice injected with HCCR-2H cells showed palpable tumors after 20days as shown in FIG. 24.

Nude mice bearing HCCR-2H allografts display characteristics of anepithelial carcinoma. FIG. 25 shows hematoxylin-eosin staining ofsubcutaneous tumor nodules taken from the nude mice. The sections of thetumor nodules revealed typical epithelial cell nests separated byfibrous stroma.

EXAMPLE 13 Establishment of New Cancer Cell Line from HCCR-2HCell-induced Tumor Tissue

The cells obtained from the tumor tissue of Example 12 was cultured in aconventional manner using 20% fetal bovine serum and the cultured cellswere designated HCCR-2HN cells which have cytological features similarto HCCR-2H cells in vitro as shown in FIG. 26.

Further, northern blotting analyses for wild type 293 epithelial celland HCCR-2-transfected 293 epithelial cells (clones A, B, C, D and E)were carried out. As shown in FIG. 27, HCCR-2 was transcribed at a highlevel in all 5 clones, while the expression of HCCR-2 gene is barelyobservable in wild type 293 epithelial cell.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. A human cervical cancer 2 protooncogene having the nucleotidesequence of SEQ ID NO:
 1. 2. A human cervical cancer 2 protooncogenehaving the nucleotide sequence comprising nucleotides Nos. 63 to 977 ofSEQ ID NO:1 and encoding the amino acid sequence of SEQ ID NO:
 2. 3. Avector comprising the protooncogene of claim
 1. 4. A microorganismtransformed with the vector of claim
 3. 5. The microorganism of claim 4,which is E. coli JM109/HCCR2(Accession NO: KCTC 0668BP).
 6. A processfor preparing a protein having the amino acid sequence of SEQ ID NO: 2comprising the step of culturing the microorganism of claim
 4. 7. A kitfor diagnosis of cancer which comprises the protooncogene of claim
 1. 8.A process for preparing a protein having the amino acid sequence of SEQID NO: 2 comprising culturing the microorganism of claim
 5. 9. A kit fordiagnosing cancer which comprises the protooncogene of claim
 2. 10. Avector comprising the protooncogene of claim
 2. 11. A microorganismtransformed with the vector of claim
 10. 12. A process for preparing aprotein having the amino acid sequence of SEQ ID NO: 2 comprisingculturing the microorganism of claim 11.